Profiling of uremic serum by high-resolution gas chromatography-electron-impact, chemical ionization mass spectrometry

Profiling of uremic serum by high-resolution gas

chromatography-electron-impact, chemical ionization mass

spectrometry

Citation for published version (APA):

Schoots, A. C., Mikkers, F. E. P., Cramers, C. A. M. G., & Ringoir, S. M. G. (1979). Profiling of uremic serum by high-resolution gas chromatography-electron-impact, chemical ionization mass spectrometry. Journal of

Chromatography, B: Biomedical Sciences and Applications, 164(1), 1-8. https://doi.org/10.1016/S0378-4347(00)81565-0

DOI:

10.1016/S0378-4347(00)81565-0

Document status and date: Published: 01/01/1979

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1

Journal of Chromatography, 164 (1979) l-8 Biomedical Applications

o Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

CHROMBEO. 372

PROFILING OF UREMIC SERUM BY HIGH-RESOLUTION GAS

CHROMATOGRAPHY-ELECTRON-IMPACT, CHEMICAL IONIZATION MASS SPECTROMETRY

A.C. SCHOOTS, F.E.P. MIKKERS end C.A.M.G. CRAMERS

Department of Instrumental Analysis, Eindhoven University of Technology, Eindhouen (The Netherlands)

and

S. RINGOER

Department of Medicine, NephroIogical Division, University Hospital, University of Ghent,

Ghenf (Belgium)

(Received February 26th, 1979)

SUMMARY

A fast and reliable procedure for gas chromatographic profiling of components in ultra- filtrated uremic serum has been developed, using glass capillary columns. Sample pretreatment consists of ultrafiltration, evaporation and silylation. Some twenty components are identi- fied by electron-impact and chemical ionization mass spectrometry. A comparison is made between profnes of sera from a series of uremic patients, before and after hemodialysis, and from non-uremic sera. Significant differences are found between these profiles_ A “dialysis ratio” is introduced as a parameter for the removal of retained components by hemodialysis treatment.

INTRODUCTION

Patients with endstage renal failure have to be submitted to regular treat- ment with an artificial “kidney”. These patients show a complex of clinical symptoms, usually called “the uremic syndrome”, or “uremia”. Many of. these symptoms are related to a disturbance in the homeostatic or regenerative function of the kidney, which results in retention of metabolic products’and in disorders of hormonal and metabolic function.

There are indications that retained components can act as cell toxins or as inhibitors of enzyme action. The identity of these components is still subject. to discussion. In recent years several authors have mentioned- the importance of compounds of medium molecular weight [l-3] .

inositol and other compounds [4-9]. Although experiments were carried out to test the various hypotheses, no definitive conclusions could be drawn [lo].

Therefore it is difficult to improve “artificial kidney” strategies and equip- ment in a planned and efficient way. For this purpose, it is necessary to

develop analytical techniques which give data on the effectiveness of the

treatment. Moreover, analytical information can lead to clinical tests on the

toxicological behaviour of certain compounds, and could contribute to a bet-

ter understanding of biochemical and physiological processes in uremia. Several profiling techniques have been applied in the analysis of body fhclids of uremic

patients? These profiling techniques give information for a whole range of

compounds. Dztik et al. 1111, Chang [X2], Gordon et al. [3], Cueille [13]

and others tried to characterize uremic plasma by gel permeation chromato- graphy, especially with reference to “middle molecules”. Senftleber et al. [14] and Veening 1151 analysed serum and hemodialysis fluid with reversed-phase

liquid chromatography. Mikkers et al. [l6] reported on an isotachophoretic

profiling technique which gives information on ionic compounds with low as

well as high molecular weight. Masimore et al. [17] examined volatile com-

ponents in hemodialysis fluid by gas chromatography-mass spectrometry

(GC-MS) using packed columns. Bultitude and Newham [18] applied the

same technique in the analysis of uremic serum, also using packed columns.

In the latter, a laborious and time-consuming sample pretreatment procedure

of several days, including fractionation by gel permeation chromatography

and freeze-drying, was used. In this report a reliable GC-profiling technique,

using glass capillary columns, and a fast pretreatment procedure is described. EXPERIMENTAL

Samples, reagents and materials

Blood samples of ten uremic patients, before and after hemodialysis on

polyacrylonitrile RP6 (Rhone-Poulenc, Paris, France) and cuprophane GM

(Gambro-Major, Lund, Sweden) membranes, were obtained from the Ne-

phrological Division of the University Hospital of Ghent (Belgium). After

centrifugation, serum samples were stored at -18O until used. A pool of

serum from non-uremic persons was prepared. Removal of high molecuiar

weight substances -was carried out by ultrafiltration on Amicon (Lexington,

Mass., U.S.A.) XM 50 membranes. Chemical derivatization was performed in

borosilicate reaction vessels (Hewlett-Packard, Avondale, Pa., U.S.A.) with bistrimethylsilyhrifluoroacetamide (BSTFA) from Pierce (Rockford, Ill., U.S.A.). Straight-chain C13- and C22-hydrocarbons [(from Phillips Petroleum (Bartlesville, Okla., U.S.A.) and Applied Science Labs. (State College, Pa., U.S.A.)] were used as internal standards. A standard solution was prepared by dissolving 21 mg of Cl3 and 4.9 mg of C22 in 50 ml n-hexane. Reagent

gas for chemical ionization (CI) was isobutane (CH 35) from 1’Air Liquide

3

Apparatus

Pressure-ultrafiltration was carried out in a microcell [19]. For GC separa- tions a Perkin-Elmer F-30 instrument was used. The standard sample intro- duction system was replaced by a moving-needle injector [20] _ Average carrier

gas velocity (helium) was 28 cm/set. An oven temperature programme was used starting with an isothermal period of 2 min at 110”) an increase of 5”/min

to 200” and remaining isothermic at 200 o for 35 min. Injection and detection temperatures were maintained at 250”. Glass capillary columns (47 m), deacti- vated [21] with Carbowax 20M and coated with SE-30 (layer thickness 0.2 pm) were prepared by the static coating procedure of Rutten and Rijks [22]. The flame ionizafion detector signal was recorded on two traces because of large concentration differences for different compounds (2 mV and 50 mV full scale corresponding to 4-10-12 A and 2-lo- lo A respectively)_ Mass spectra were obtained with a 4000 GC-MS system from Finnigan (Sunnyvale, Calif., U.S.A.) coupled to a D 116E minicomputer (Digital Computer Controls). A platinum-iridium capillary was used as a GC-MS interface. In MS analysis the electron energy was 70 eV in both the electron-impact (EI) and the CI mode. Source temperature was 250” under EI and 220” under CI, sensitivity 10-g and 10Wy A/V, multiplier voltage 1675 V, .and reagent gas pressure 13 Pa (0.1 Torr.) in chemical ionization.

Procedure

Serum samples are pressure-ultrafiltrated under nitrogen to remove high molecular weight components such as proteins (cut-off at ‘50,000). Aliquots of 250 ,ul of the ultrafiltrated material are evaporated to dryness under a ni- trogen stream in a sandbath at 70” _ The dried samples are derivatized to enable

GC separation and are allowed to react with 250 yl of BSTFA reagent at 80” for 2 h. After dilution with 250 ~1 n-hexane, aliquots of 50 ,ul of the standard solution (Cl3 and C22 in hexane) are added.

Samples of 0.5 J.L~ are applied to the tip of the moving needle. After 90 set, during which solvent, volatile reaction products and unreacted BSTFA are allowed to evaporate, the sample is injected.

RESULTS AND DISCUSSION Reliability of the method

As a retention parameter the relative retention is used. In our experiments

this parameter appeared to be more reliable than the retention index. The

reproducibility of the temperature programme was tested by injecting the same sample six times within a short period. Five peaks (peak numbers 35, 49, 67, 75, 78 in Fig. 1) throughout the whole temperature range show coef- ficients of variation for the relative retention (with respect to C22) which are less than 0.4%. This demonstrates that the reproducibility of the tempera- ture programme is good.

The same peaks were tested in chromatograms that were recorded in the course of two months. Coefficients of variation between 0.2 and -2% for dif- ferent peaks represent long-term changes in column performance and carrier gas flow. This result is satisfactory, however, the maximum value of 2% can

cause difficulties in distinguishing one peak from another for certain compo- nents (e.g. peaks 34 and 35 in Fig. 1).

Peak height is

used

as

unresolved peaks this gives better results than peak area measurement. Repro- ducibility of the normalized peak heights, from four injections, is measured for

15

different peaks were between 2 and 9%. The influence of the sample pretreat- ment on peak height variation was studied by derivatizing an ultrafillrated serum sample four times. Each sample was then injected four times. A Stu- dent’s t-test (on the mean) and a F-test (on variance ratio) were applied to peak heights “within and between” samples. This led to the conclusion that variance due to sample pretreatment does not differ from variance from the analysis step (95% probability level)_

Application to a series of uremic patients

Predialysis and postdialysis serum samples were submitted to the described procedure. Typical gas chromatograms are shown in Fig. 1. Differences in

Fig. 1. Gas chrornatographic profiles of ultrafiltrated serum from an uremic patient, before and after bemodialysis treatment, and from a pool of non-uremic sera. Glass capillary column coated with SE-30. The 2-mV trace corresponding to a signal of &lo-” A f.s.d is shown.

TABLE I

CLASSIFICATION ACCORDING TO DIALYSIS RATIO

Group 1 2 3 4 - DX 0.6dD < 1.4 D> 1.4 D < 0.6 variating Number of peaks 8 36 1 13 Major GC peabii 22, 27, 28, 67,B l8, 24, 26, m, 30, 32 33 34,& 36, -t -, *a, 42, 43, 46, &9, 53, 54, 54a,.58,62a, 72 73 74, B _, -. Ss 23, 44, 55, 61, 62, 63 66 76a -*-*_

concentrations of various components are very obvious. All ten patients showed similar profiles, however, individual deviations, both qualitative and quantitative, did occur. For quantification of the effect of hemodialysis treat- ment a “dialysis ratio” (D) is defined in the following way:

H’ x, before D,=

Et x, after

where HX is the normalized peak height for component X.

This dialysis ratio (D) was determined for some 70 peaks in the chromato- grams of the ten patients_ From these data an average D (B) was calculated for each compound. Then the components were classified into four groups, ac- cording to their D. The maximal error in D can be calculated from that in peak height. A maximal variation coefficient of 9% in peak height was found. This leads to a maximum value of approximately 20% for the variation coefficient (V.C.) of D. On this basis all components with D-values between l-O.4 and 1+0.4 were considered to be unaffected by hemodialysis treatment (D f 2 V.C.). These components are classified in group 1 (see Table I). Group 2 re- presents components that show a decreased concentration as a result of dialysis treatment (Da1.4). Only very few components showed higher concentrations after hemodialysis, and are placed in group 3 (D<O.6). Group 4 represents components that show a great variation ;J1 D in the samples of different patients. The table demonstrates that group 2 components are very well re- moved during dialysis, and that concentrations of group 3 components appear to be raised as a result of the treatment. From peak heights and D-values for samples of different patients it is concluded that the higher the concentrations, the higher the dialysis ratio. The ten patients show substantial differences in ‘coverall” concentrations. Some of them have postdialysis profiles that ap- preach those for non-uremic sera.

The underlined peak numbers in Table I refer to compounds that identified by GC-MS and from GC retention data. Table II lists these pounds.

were com-

TABLE II

IDENTIFIED COMPONENTS IN UREMIC SERUM

Peak Relative number retention Compound* Dialysh ratio (D) Group 9 0.153 10 0.178 11 0.183 18 0.228 22 0.251 26b 0.296 30 0.322 32 0.327 33 0.336 35 .0.352 40a 0.403 42 0.417 49 0.469 54/54a 0.498/0.504 55 0.507 56 0.520 58 0.530 61 0.553 63 0.574 66 0.591 67 0.605 72173 0.665/0.682 74 0.695 75 9.716 76a 0.751 78 0.900 urea phosphoric acid glycerol

tartronic acid (tent.) threonine homoserine (tent.) A-pyrrolidone-5carboxylic acid threitol (tent.) erythritol erythronic acid tartaric acid 2-deoxy-erythropentonic acid arabinitol

hydroxy or dicarboxylic acids arabinonic acid citric acid fructose galactose 3-deoxy-arabinohexonic acid (tent.) glucono-1,4-lactone 05D-&cos~

mannitol and/or glucitol isomer of myo-inositol (tent.) fl-D-glucose

mannonic or gluconic acid myo-inositol 1.40 2.40 - 2.71 1.05 1.47 2.70 2.30 2.10 3.27 3.42 1.96 2.93 2.9813.24 - 0.57 1.61 - - 2 2 - 2 1 2 2 2 2 2 2 2 2 212 4 3 2 4 4 - 1.25 6.0913.72 3.52 1.28 - 7.00 4 1 212 2 1 4 2 “Tent. = tentatively identified.

Identification by mass spectrometry

Many carbohydrate-related trimethylsilyl derivatives have EI mass spectra that look very similar. Although different classes of these compounds (e.g. aldoses, alconic acids and poiyols) demonstrate some characteristic fragment or rearrangement ions, no molecular ions are found [23-261. Because of this similarity, reference spectra from different origin were used [24, 25, 271. Moreover extra information on molecular weight was obtained for some com- ponents by recording CI mass spectra. Although polyols and aldonic acids showed molecular ions in these spectra, aldoses did not. Differences between EI and CI spectra for several classes of compounds will be discussed in a separate publication. Table III shows the highest mass ions in EI and CI (iso- butane) spectra of some components.

Peak 16 (Fig. 1, Table II) must be a hydroxy acid or a dicarboxylic acid with molecular weight of 336. Its spectrum (EI) shows an abundant peak at

m/z 292, which probably results from a McLafferty-type rearran gement of a trimethylsilyl group [28]. The m/z 292 ion is the highest mass ion in the EI spectrum. The CI spectrum shows a peak at m/z 337, which is probably the (M+l)‘ molecular ion. Peak 18 is therefore tentatively identified as tartronic

7 TABLE III

COMPARISON OF EI AND CI SPECTRA Peak

number Compound name Mol.

Highest mass ions in

weight EI (70 eV) CI (70 eV, 13 Pa)

33 35 40a 42 49 56 78 erythritol4TMS erythronic acid-4TMS tartaric acid-4TMS 2-deoxyerythropentonic acid-4TMS arabinitol-5TMS citric acid-4TMS inositol-6TMS 410 320,307,293,277 411,321,305,293 424 409,379,319,292 425,409,335,307 438 423,333,305,292 439,423,321,292 438 348,335,333,321 439,423,349,333 512 320,319,317,307 513,333,307,303 480 465,375,363, 347 481,465,363,319 612 507,432,393,367 613,433,393,367

acid (hydroxymalonic acid)_ The spectra of peaks 54 and 54a also show ions at m/z 292, but no molecular weight information is available. Hippuric acid, which can not be derivatized in a reproducible way, eluted in a few chroma- tograms simultaneously with arabinonic acid (peak 55). Peak 74 shows an EI spectrum that is similar to the spectrum of myo-inositol (peak 78). More- over peak heights of peaks 74 and 78 seem to be related to each other, so it is to be concluded that it is an isomer of myo-inositol.

The components at peak numbers 22, 26b, and 30 were included in Table II at the last moment. Obviously they are related to amino acid metabolism. Peak 30 was identified as A-pyrrolidone-5carboxylic -acid. This is a product of an intramolecular peptide bonding (cyclisation) in glutamic acid. It is not known whether this compound is really present in uremic serum in this quanti- ty or is formed from glutamic acid in the derivatization step [29] .

CONCLUSIONS

A reproducible and reliable GC method for profiling of uremic serum has been described. Profiles from pre- and postdialysis serum show that hemo- dialysis treatment results in a significant decrease of the concentration of many components. However, it is observed that different components are not removed to the same extent. Therefore a component-specific parameter, the dialysis ratio, is introduced. It could be seen that different patients showed substantial differences in “overall” concentration. Some patients showed postdialysis profiles that seemed “worse” than predialysis profiles of other patients. Some postdialysis profiles had “overall” concentrations comparable to those for non-uremic serum. Components that are detected by this method

are related to carbohydrate metabolism, such as aldoses, aldonic acids and polyols. Also other organic acids and some nitrogen containing compounds are detected. The toxicological behaviour of these components is not yet well understood [4].

In order to include other classes of compounds it is necessary to apply

several techniques simultaneously. The isotachophoterid. profiling technique

developed in this laboratory by Mikkers et al. [16], is very suitable for pro-

filing of ionic substances in serum.

pretreatment ‘procedure of several days including fractionation by gel per-

meation chromatography. Despite the fact that no such fraction technique

was applied in our GC method, the same range of compounds (and some

more) are detected.

The use of glass capillary columns gives more detailed information than

earlier investigations using packed columns. ACKNOWLEDGEMENTS

Ii-. G.A.F.M. Rutten is gratefully acknowledged for preparing the excellent

GC cohunns. We also thank Ir. G.A.F.M. Rutten, Dr. J.A. Rijks and Dr. P.A.

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